The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of Streptococcus pneumoniae infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This phenomenon has created a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.
N-Acetylglucosamine-1-Phosphate Uridyltransferase (GlmU) catalyses the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan in all bacteria and of lipopolysaccharide and enterobacterial common antigen in gram negatives. The enzyme has been purified from Escherichia coli and is bifunctional, also catalyzing the preceeding step of N-acetylation of glucosamine-1-phosphate (Mengin-Lecreulx, D. and van Heijenoort, J, J.Bacteriol. 176: 5788-5795 [1994]). It is possible to block the acetyltransferase activity but not the uridyl transferase activity with thiol inhibitors, suggesting that the enzyme may have two domains. The gene, glmU, encoding the enzyme has been cloned from E. coli (Mengin-Lecreulx, D. and van Heijenoort, J, J.Bacteriol. 175: 6150-6157 [1993]) and its counterpart in Bacillus subtilis (gcaD) has also been identified (Hove-Jensen B, J.Bacteriol. 174: 6852-6 [1992]).
The essential nature of the gene product of gcaD is demonstrated by temperature sensitive mutants of Bacillus subtilis which are unable to make active enzyme and stop growing at the restrictive temperature (Hove-Jensen [1992]). Inhibitors of these proteins therefore have utility in anti-bacterial therapy. The discovery of the gene from the human pathogen Streptococcus pneumoniae corresponding to gcaD/GlmU permits production of the enzyme which can be used to screen for novel antibiotics.
Clearly, there exists a need for factors, such as the GlmU embodiments of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess amino acid sequence homology to a known GlmU protein from Bacillus subtilis. See PIR database S66050; Genembl D26185; and Swissprot P14192. Also see NILSSON D., HOVE-JENSEN B., ARNVIG K. MOL. GEN. GENET. 218:565-571 (1989); OGASAWARA N., NAKAI S., YOSHIKAWA H. DNA RES. 1:1-14 (1994).